Immunization of MERS-CoV-2-infected mice with a sublethal dose of MERS-CoV or VRP-MERS-S

Summary

Here, we detail the immunization of mice with a sublethal dose of MERS-CoV or two doses of replication-incompetent alphavirus replicon particles expressing MERS-CoV spike protein. We then describe steps to determine the outcome of immunization by challenging immunized mice with a lethal dose of MERS-CoV, as well as by detecting virus-specific neutralizing antibody and virus-specific T cell response via neutralization assay and flow cytometry, respectively. This protocol can be used to evaluate other CoV infections or vaccine-induced immune responses.

For complete details on the use and execution of this protocol, please refer to Zheng et al. (2021).¹

Graphical abstract

Highlights

• •Mouse model to determine MERS-CoV-specific immune responses
• •A replication-incompetent alphavirus VRP-MERS-S is produced to immunize mouse model
• •Protective effects induced by immunization are evaluated via in vivo and ex vivo systems
• •Virus-specific T cells are determined by flow cytometry following peptide stimulation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we detail the immunization of mice with a sublethal dose of MERS-CoV or two doses of replication-incompetent alphavirus replicon particles expressing MERS-CoV spike protein. We then describe steps to determine the outcome of immunization by challenging immunized mice with a lethal dose of MERS-CoV, as well as by detecting virus-specific neutralizing antibody and virus-specific T cell response via neutralization assay and flow cytometry, respectively. This protocol can be used to evaluate other CoV infections or vaccine-induced immune responses.

Before you begin

Pathogenic human coronaviruses, which include severe acute respiratory syndrome-coronavirus (SARS-CoV), Middle East respiratory syndrome-CoV (MERS-CoV), and SARS-CoV-2, cause mild to lethal respiratory symptoms and systemic complications.² Despite the detrimental outcomes resulting from dysregulated immune responses characterized by hyperinflammation (cytokine storm), the host immune system plays a crucial role in restricting the entry, replication, and transmission of virus.^3,4

While SARS-CoV-2 has caused a global pandemic, MERS-CoV has been identified by the World Health Organization (WHO) as a priority pathogen and by the Coalition for Epidemic Preparedness Innovation (CEPI) as a candidate for vaccine development because it also causes severe disease in humans and has epidemic potential. Currently, no therapeutic options are available to treat MERS-CoV.⁵ Mice are normally resistant to infection with MERS-CoV but are rendered susceptible to the virus if the human receptor dipeptidyl peptidase-4 (hDPP4) is present. To assess MERS-CoV infection outcomes in experimental animals, we generated mice knocked-in for hDPP4 (hDPP4-KI mice) and then further adapted the virus to mice by serial passages through these mice.⁶ These mice were immunized with a sublethal dose of MERS-CoV or with two doses of replication-incompetent alphavirus replicon particles expressing MERS-CoV spike protein (VRP-MERS-S), followed by challenge with a lethal dose of MERS-CoV.¹

In addition to assessing protection in mice, we also evaluated the humoral and cellular immune memory response. Since virus-specific neutralizing antibodies are critical for protection against re-challenge with homologous virus,^7,8,9,10 a plaque reduction neutralization test (PRNT₅₀) is used to determine the titer of serum virus-specific neutralizing antibodies in this protocol. T cell-mediated immune memory also plays a crucial role in protecting the host from challenge by virulent pathogens,^{11,12,13,14,15} which was further highlighted by the occurrence of antibody-resistant SARS-CoV-2 variants of concern (VOCs).^10,16,17,18 In this protocol, lung and spleen cells harvested from immunized mice were stimulated with MERS-CoV-derived peptides or peptide pools ex vivo, followed by measuring interferon (IFN)-γ, tumor necrosis factor (TNF) and interleukin (IL)-2 expression of T cells through flow cytometry assay. Alternative methods to detect immune memory responses induced by infection or vaccines are discussed in expected outcomes and limitations.

The protocol below describes the specific steps needed for immunizing mice with a sublethal dose of live MERS-CoV or two doses of VRP-MERS-S, followed by the determination of memory T and antibody responses upon stimulation with homologous virus or viral antigens. In addition, we have also applied a similar strategy to induce and detect SARS-CoV and SARS-CoV-2-specific immune memory responses using corresponding virus, VRP, and virus-specific peptides/peptide pools.

Institutional permissions

All animal studies described here were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Iowa and followed the Guide for the Care and Use of Laboratory Animals. The readers also need to obtain institutional permission for all animal work.

MERS-CoV propagation and titration

Timing: 4 weeks

• 1.
  Preparation of Vero 81 cell (Week 1) (The following procedures need to be carried out in Biosafety Level 2 cabinet under institutional permissions).

  • a.
    Preparation of culture medium.

    • i.
      Thaw Fetal bovine serum (FBS, s11150, Atlanta Biologicals) in a 4°C refrigerator for 16 h (hr) followed by inactivation in a 56°C water bath, 30 min (min).

      Note: After inactivation, FBS can be kept in 4°C refrigerator for 1 month or aliquoted and kept in −20°C freezer for 6 months. Avoid repeated thawing and freezing FBS.

    • ii.
      Mix 60 mL inactivated FBS with 500 mL DMEM (glucose and antibiotics-free, 11960-044, GIBCO).
    • iii.
      Add 6 mL 10,000 Units/mL Penicillin/10,000 μg/mL streptomycin (15140-122), 4 mL 100 mM sodium (Na) pyruvate (11360-070), 6 mL 100× non-essential amino acid (NEAA) (11140-050), 12 mL 7.5% Na-bicarbonate (25080-094), and 12 mL 200 mM L-glutamine (25030-081) into the mixture to make complete 10% FBS-DMEM (D10) medium.

      Note: all reagents used in this protocol are purchased from GIBCO, although alternatives from other companies may be used as alternatives.

      Note: The preparation of medium is carried out in a biosafety cabinet.

    • iv.
      Pre-warm 12 mL D10 in a 37°C water bath before use.
  • b.
    Thaw 1 vial of Vero 81 cell (stored in liquid nitrogen) in a 37°C water bath and add it immediately by pipetting into a 12 mL pre-warmed D10 medium using a 1000 μL pipetman.
  • c.
    Count cells using a hemacytometers under a microscope (10× objective).
  • d.
    Transfer 2–5×10⁶ cells (in 12–15 mL D10 medium) into a T-75 ventilated-cap flask (treated for increased cell attachment, 10861-646, Avantor) with a 10 mL serological pipet and culture cells in a 37°C incubator containing 5% CO₂.
  • e.
    Monitor the confluence of Vero 81 under microscope daily and split the cells when they reach 90% confluency (characterized by scatter blank area with no overlay of cells), which usually takes 2–3 days.

    • i.
      Remove the medium from T-75 flask to a vacuum waste bottle by aspiration and rinse cells with 10 mL pre-warmed 1× PBS (10010-023, GIBCO) using a 10 mL serological pipet.
    • ii.
      Add 2 mL 0.25% Trypsin-0.53 mM EDTA (25200-056, GIBCO) into the flask and place the flask in a 37°C incubator for 5 min.
    • iii.
      Add 10 mL of D10 into the flask to stop the enzymatic activity of EDTA-Trypsin when cells round up under microscope and before they detach from the flask surface.
    • iv.
      Gently pipette Vero 81 cells off the flask surface with a 10 mL serological pipet.
    • v.
      Split the cells at 1:4–1:8 ratio with fresh D10 medium, 12–15 mL/T-75 flask.
      Or aliquot the cells into 12-well tissue culture plates (Corning), 2 mL/well at a concentration of 0.25×10⁶/mL, for virus titering.
    • vi.
      Place flasks or plates in a 37°C, 5% CO₂ incubator.

      Note: The cells in the flasks can be split or used for infection when they reach 90% confluence. The plates will be used for titering when the confluence of cells reach 100%, which usually take 12–24 hr.

• 2.
  MERS-CoV propagation and titration (Week 2) (All of the following procedures need to be carried out in a BSL3 Lab with institutional permissions. Persons working with MERS-CoV need to wear institutional biosafety committee approved personal protective equipment (PPE). All waste generated in this protocol must be inactivated before discarding following institutional guidelines).

  • a.
    Propagation of MERS-CoV:

    • i.
      Remove the Vero 81 cell medium to a vacuum waste bottle by aspiration and rinse cells with pre-warmed PBS.
    • ii.
      Calculate the amount of virus to be used:
      Infection dose: multiplicity of infection (MOI) = 0.01.
      Cell number: a 90% confluent T-75 flask contains ∼10×10⁶ Vero 81 cells.
      Virus to be used = MOI × Cell number = 10⁵ PFU/ T-75 flask.
    • iii.
      Thaw 1 vial of MERS-CoV (EMC2012 strain, stored in −20°C freezer) in a 4°C refrigerator or on ice.

      Note: The virus needs to be kept on ice during the whole procedure. Dilute virus with cold serum-free DMEM to a concentration of 5 × 10⁴ PFU/mL.

    • iv.
      Remove PBS from Vero 81 flasks into the waste bottle by aspiration and add virus-containing medium into flasks, 2 mL/ T-75 flask.
    • v.
      Return the inoculated flasks to the 37°C incubator. Adsorb the virus to cells for 1 h by gently shaking flasks every 15 min.
    • vi.
      Add 10 mL 2% FBS-DMEM (D2)/flask and return the flasks to the 37°C, 5% CO₂ incubator.
    • vii.
      Monitor inoculated flasks every 12 h and remove flasks to a −80°C freezer when virus-induced cytopathic effects (CPE) are observed in 25% of cell-attached area (Figure 1). This usually takes 48–72 h.

      Note: The flasks need to be sterilized by wiping with approved reagent (such as Virex (Virex II 256), 2% MicroChem (NCL, 1839-95-2296), or alternatives according to institutional guidelines) and sealed in a secondary container (such as ziplock bag or air-tight plastic box) before moving into the freezer. The flasks can be kept in −80°C freezer for 24hr to months per users’ convenience.

    • viii.
      Thaw frozen flasks in a 4°C refrigerator (it usually takes 2 h) and gently pipette off the rest of attached cells with a 10 mL serological pipet.
    • ix.
      Pool harvested virus-containing medium into 15 mL or 50 mL tubes, spin at 300× g, 10 min, in a 4°C pre-cooled centrifuge to remove cellular debris.
    • x.
      Carefully transfer the supernatant into new 15 mL or 50 mL tubes without disturbing the pelleted debris.

      Optional: Filtration of harvested supernatant with a 0.25–0.5 μm filter (Corning) may be used to further clarify the supernatant.

      Note: The loading and unloading of centrifuge tubes need to be carried out in a Biosafety cabinet. After centrifugation, the tubes need to be rested vertically for 2–3 min before opening, to avoid the spread of aerosols created by centrifuging.

    • xi.
      Aliquot virus-containing supernatant into 2 mL screw cap tubes (0.05–1.5 mL/tube), as desired.
    • xii.
      Label tubes with the name of preparer, date, and virus strain.

      Note: Store aliquots in a −80°C freezer for future use. The titers of CoV are stable at −80°C for 2 years.

  • b.
    Titration of cultured MERS-CoV by the plaque assay.

    • i.
      Prepare 2–3 12-well plates of Vero 81 (100% confluence) as described in MERS-CoV propagation and titration.
    • ii.
      Thaw 1 vial of aliquoted MERS-CoV in a 4°C refrigerator or on ice and make serial dilutions using cold serum-free DMEM.

      Note: The virus should be kept on ice during the whole procedure. 10× serial dilutions are usually applied for the first titration. 2× or 4× serial dilutions can be used to determine the titer more accurately.

    • iii.
      Remove the medium from Vero 81 plates to a waste bottle by aspiration and rinse cells with pre-warmed 1× PBS, 2 mL/well.
    • iv.
      Remove PBS by aspiration and add diluted virus into plates, 0.2 mL/well (make triplicates for each dilution).

      Note: Always leave 1 column (3 wells) for negative controls (0.2 mL virus-free DMEM/well) and 1 column for positive controls (0.2 mL titrated virus/well) (An example is shown in Table 1).

    • v.
      Return plates to the 37°C incubator, and adsorb the virus for 1 h by gently shaking plates every 15 min.
    • vi.
      Remove the virus-containing medium to a waste bottle containing bleach (or alternative inactivating solutions according to institutional guidelines) by aspiration and rinse cells with pre-warmed 1× PBS, 2 mL/well.
    • vii.
      Mix 2× DMEM (powder is purchased from GIBCO, 12100-046, and 2x DMEM is prepared by the investigator using double-distilled water (ddH₂O)) and 1.2% agarose (powder is purchased from Roche, 11685678001, and the solution is prepared by the investigator using 1× PBS) at a ratio of 1:1 to make 0.6% agarose in DMEM immediately before usage.

      Note: 1.2% agarose will solidify under 40°C. We usually store prepared agarose at 25°C and melt it using a microwave before usage. To avoid the re-solidification of 1.2% agarose, it needs to be placed in a 65°C water bath until mixing with 2× DMEM.

    • viii.
      Remove PBS by aspiration and quickly add 0.6% agarose-DMEM solution into plates, 0.5 mL/well.
    • ix.
      Leave plates in the Biosafety cabinet at 25°C, until the agarose solidifies.
    • x.
      Add 0.5 mL/well D2 to overlay the agarose-DMEM gel.
    • xi.
      Return plates to the 37°C, 5% CO₂ incubator.
    • xii.
      72 h later, remove D2 medium and add 0.5 mL 10% Formalin/well (SIGMA, HT501128) to fix cells at 25°C for 15 min.
    • xiii.
      Remove 10% Formalin and agarose-DMEM gel.

      Note: The agarose-DMEM gel needs to be removed carefully using forceps without disrupting the cell layer, which requires practice.

    • xiv.
      Add 0.2 mL 0.1% Crystal violet (Powder is purchased from Fisher, C581) into each well at 25°C for 5 min.

      Note: 0.1% Crystal violet: 5 mL 1% Crystal violet with 45 mL 1×PBS, prepared immediately before usage.

    • xv.
      Remove Crystal violet solution and read plaques in each well (Figure 2).

      Note: After staining with Crystal violet, the plates can be stored at 25°C for extended periods of time (months). Before moving out of the Biosafety cabinet, the plates need to be sterilized by wiping with approved reagent.

    • xvi.
      The titer of virus (plaque forming units, PFU) is calculated as following:

      $$PFU=\frac{Averageplaquesoftriplicates\times foldsofdilution}{infectionvolume}$$

      Note: The titration of MERS-CoV may range from 10² to 10⁸ PFU/mL. We usually use samples in which plaques can be clearly delineated and average the titer across dilutions. Do not use wells containing merged plaques or >50 plaques to calculate the titer, because quantification will be inaccurate.

• 3.
  Assess MERS-CoV virulence in mice (Week 3 and 4) (All of the following procedures need to be carried out in an Animal Biosafety Level 3 (ABSL3) Lab wearing personal protective equipment (PPE)). All manipulations must be reviewed and approved by the Institutional Animal Care and Use Committee).

  • a.
    Preparation of hDPP4-KI mice.

    • i.
      hDPP4-KI mice are maintained in a specific pathogen-free (SPF) animal facility at the University of Iowa. Mice are weaned at 21–28 days.
    • ii.
      Animals will be acclimatized in the ABSL3 for 3–5 days before infection.
    • iii.
      Drinking water and bedding are replaced every 1 week and 2 weeks respectively. Extra interactions should be avoided to reduce the stress of mice.
    • iv.
      Before infection, the general condition (posture, fur status, activity, intake of food and water) and the weight of mice are monitored and recorded daily. Mice showing abnormal symptoms (hunch, irritability, reduced activity, weight change and reduced intake of food and water) are excluded or euthanized.
  • b.
    Infection of hDPP4-KI mice.

    • i.
      8-10-week-old male or female hDPP4-KI mice are randomly distributed into different groups. The infectious doses will be determined based on prior results or published references.

      Note: Male hDPP4-KI mice are usually more susceptible to MERS-CoV infection compared to their age-matched female littermates.

    • ii.
      Thaw 1 vial of MERS-CoV in a 4°C refrigerator and make serial dilutions with cold serum-free DMEM.

      Note: The diluted virus should be kept on ice during the whole procedure.

    • iii.
      Anesthetize mice with 100 mgkg⁻¹/10 mgkg⁻¹ Ketamine/Xylazine in 0.1–0.25 mL volume via intraperitoneal (i.p.) injection using a 1 mL insulin syringe (fixed needle, BD).

      Note: Anesthetized mice should be kept on a warm pad to maintain body temperature. Eye ointment will be applied to avoid dryness. Examine the anesthesia of mice every 2 min by touching or pinching the tail or foot of mice. Manipulations need to be carried out under full anesthesia (e.g., no response to tail or limb pinch) to reduce pain and stress of animals.

    • iv.
      Hold the mouse at an upright position and inoculate diluted virus into nostrils using a 100 μL pipetman, 25 μL /nostril, 50 μL total.

      Note: Inocula are given in small drops within 15–30 s (sec). Manipulation should be paused if respiration becomes abnormal or bubbles form at the nostrils. After inoculation, hold the animal in the upright position for at least 30 s to avoid aspiration.

    • v.
      Rest mice on the warm pad until they are awake and resume activity.
    • vi.
      Return mice to original cages after complete awaking from anesthesia and monitor the weight and the general condition 1–2 times per day.

      Note: Infected mice may start to lose weight from 2 days post infection (dpi) and show systemic and respiratory symptoms (irritability, hunching, reduced responsiveness to touch, decreased normal activity, decreased intake of water and food, and respiratory stress) starting at 4–5 dpi. Liquid (Nepa Nectar) and food will be placed at the bottom of the cages for mice to reach easily. I.p. injection of 0.5 mL PBS may be necessary to treat dehydration (characterized by weakness, trouble gripping the cage bars with forefeet, sunken or recessed eyes, fuzzy facial fur). Mouse death mostly occurs between 7–10 dpi. Euthanasia will be performed when mice reach humane endpoints according to the protocol approved by the IACUC.

Figure. 1.

CPE of MERS-CoV-infected Vero 81 cells

Vero 81 cells (in T-75 flasks, 90% confluent) are infected with mock virus or MERS-CoV at MOI = 0.01 in 2 mL serum-free DMEM, 37°C. The flasks are gently shaken every 15 min. After 1 h, 13 mL 2% FBS-DMEM are added into each T-75 flask. The cells are monitored every 12 h. CPE is shown in the right panel (MERS-CoV-infected cells) at 60 h post infection. (40× magnification).

Table 1.

An example of plaque assay outcome obtained in a 12-well plate culture

NO. Of plaques	106 dilution	107 dilution	108 dilution	Negative control
	Column 1	Column 2	Column 3	Column 4
Row 1	18	2	0	0
Row 2	25	2	0	0
Row 3	29	3	0	0

PFU of virus shown above = ((2 + 2+3)/3)×10⁷/0.2 = 1.33 × 10⁸ PFU/mL (Column 2).

Or = ((18 + 25+29)/3)×10⁶/0.2 = 1.2 × 10⁸ PFU/mL (Column 1).

Figure. 2.

The outcome of plaque assay

Vero 81 cells (in 12-well plates, 100% confluent) are infected with mock virus or serial-diluted MERS-CoV in 0.2 mL serum-free DMEM, 37°C, 1 h. The plates are gently shaken every 15 min. After incubation, mock or virus-containing medium are removed, and 0.5 mL/well 0.6% agarose in DMEM are added into each well. After agarose solidifies, 0.5 mL/well 2% FBS-DMEM is added to overlay the gel. 3 days later, the media are removed, and the plates are fixed with 10% formalin, RT, 15 min. After fixation, the formalin and the gel are carefully removed and the plaques (arrows) are visualized by 0.1% Crystal violet staining, RT, 5 min.

The humane endpoints used in this protocol include:

weight loss >30% of initial weight, severe respiratory distress (wheezing, coughing, or open mouth breathing), neurologic symptoms (i.e., seizures, hind limb paralysis), unresponsive to external stimuli.

• • • vii.
      All surviving animals will be euthanized at 14 dpi by CO₂ inhalation followed by cervical dislocation, although some mice may be euthanized at different time points for tissue harvesting, which will be described in the step-by-step method details.

VRP-MERS-S preparation

Timing: 2 weeks

• 4.
  Production of VRP (All of the following procedures need to be carried out in a Biosafety Level 2 cabinet wearing personal protective equipment (PPE)).

  • a.
    Obtain pVR21 containing VEE sequences plus MERS-CoV spike protein gene but minus capsid and E glycoprotein (pVR-21-MERS-S), 3526 capsid helper plasmid, and 3526 E glycoprotein helper plasmid (Day 0) (pVR-21 and plasmids encoding the 3526 capsid and E glycoproteins were provided by Dr. Ralph Baric, University of North Carolina). (Figure 3).¹⁹

    • i.
      Digest pVR21-MERS-S with Not I enzyme (300 Unit/mL, ER0591, Thermoscientific) (Reaction system: 6.5 μL Buffer, 0.7 μL 100× bovine serum albumin (BSA), 2. 0 μL Not I, 5.8 μL H2O, 50.0 μL plasmid DNA (pDNA) at 37°C for 1 h.
    • ii.
      Clean up the digested pDNA using a PCR Product Clean Kit (Qiagen, 28104) according to the manufacturer’s manual.
    • iii.
      Transcribe RNA using Ambion T7 Transcription Kit (AM1334, Reaction system: 7.5 μL digested pDNA, 3.0 μL 10× buffer, 15 μL 2× NTP/CAP, 3 μL enzyme, 1.5 μL GTP), 37°C, 2 h.

      Note: The product can be stored at −80°C until use.

      Note: Always aliquot at least 3 μl product in an independent tube and store it at −80°C for gel analysis of the RNA (target RNA: 5kb).

  • b.
    Electroporate BHK cells for VRP production (Day 1).

    • i.
      Culture BHK cells with Minimum Essential Medium (MEM)-based medium (GIBCO, M200500).
    • ii.
      Trypsinize cells with 2 mL 0.25% Trypsin-EDTA when BHK reaches 80% confluence in a T-75 flask, 5 min at 37°C.
    • iii.
      Harvest trypsinized cells by pipetting and wash with 50 mL cold 1× PBS, centrifuge at 300× g, 10 min at 4°C.
    • iv.
      Remove the supernatant to a waste bottle by aspiration and re-suspend cells with cold 1× PBS at 1.5×10⁷/mL by counting under microscope.
    • v.
      Add 0.8 mL of cell suspension into a 0.1 cm cuvette, and rest at 25°C.
    • vi.
      Add RNA product to the cuvette and apply 3 pulses per cuvette (0.3–0.4 msec, 1.8 kV).
    • vii.
      Rest cells at 25°C for 10 min before transfer into a new T-75 containing 22 mL pre-warmed complete MEM medium. Incubate cells in a 37°C, 5% CO₂ incubator for 24 h.
    • viii.
      Collect medium by pipetting and centrifuge at 500× g for 15 min at 4°C. Store the supernatant in a 4°C refrigerator (Day 2).
    • ix.
      Add 14 mL fresh complete MEM medium into the cell-culture flask. Return the flasks to a 37°C, 5% CO₂ incubator and culture for 24 h.
    • x.
      Collect medium and centrifuge at 500× g for 15 min at 4°C (Day 3).
    • xi.
      Pool the clarified medium for VRP purification.

      Note: Always reserve at least 4 mL clarified medium in an independent tube at 4°C for safety testing by 2-rounds of BHK cell infection. CPE should only be identified after the first but not the second passage.

  • c.
    Purification and titration of VRP (Day 4).

    • i.
      Add 5 mL 20% sterile sucrose (powder is purchased from Invitrogen, 15503022, and the solution is prepared by the investigator using ddH₂O followed by autoclaving) to the bottom of each SW28 ultra clear ultra-centrifuge tube (BD) and slowly overlay with 32 mL clarified medium. Centrifuge at 10,000× g for 3 h at 4°C.
    • ii.
      Discard the supernatant by aspiration and blot tube rim on a Kimwipe (or alternative tissue paper) to remove the residual liquid.
    • iii.
      Re-suspend pellets with 0.5 mL cold 1× PBS and remove to a new tube. Rinse the tube with 0.5 mL cold 1× PBS and pool with re-suspended VRP.

      Note: Store aliquoted VRP in the −80°C freezer.

    • iv.
      Plate BHK cells on 8-well chamber slides at a concentration of 1.0×10⁴/mL, 0.2 mL/well. Let cells adhere for 16hr in a 37°C, 5% CO₂ incubator.
    • v.
      Make serial dilutions of VRP using 1× PBS (Mg⁺⁺ and Ca⁺⁺, GIBCO, 14040-133).
    • vi.
      Remove the medium from slides and add 80 μL diluted VRP/well. Gently tap the slides to guarantee that the diluted VRP covers the whole well.
    • vii.
      Place the slides in a 37°C, 5% CO₂ incubator, 1 h.
    • viii.
      Add 0.3 mL fresh complete MEM medium into each well and incubate for 18–24 h.
    • ix.
      Remove the medium and cover cells with 0.2 mL 1:1 mixture of acetone (SIGMA, 270725) and methanol (SIGMA, 34860) at 4°C to fix it for 1 h.
    • x.
      Re-hydrate cells with 1× PBS (Mg⁺⁺ and Ca⁺⁺) at 25°C, 15 min.
    • xi.
      Rinse slides with 1× PBS twice and culture with mouse anti-MERS-CoV sera (prepared from MERS-CoV-infected mice and verified by PRNT₅₀ assay by our lab, 1/200) at 25°C, 1 h.
    • xii.
      Rinse slides with 1× PBS three times and cover with FITC-labeled goat anti-mouse IgG (1/50) at 25°C, 30 min.
    • xiii.
      Rinse slides with 1× PBS three times and dry the slides using KimWipes or alternative tissue paper.
    • xiv.
      Cover cells with anti-fade buffer (Vector Laboratories, UX-93952-24) and count the number of positive (fluorescence of FITC) cells under a fluorescent microscope.
    • xv.
      The titer of VRP is calculated as following:

      $$\frac{VRP}{ml}=\frac{\left(\frac{averagegreencellcountsperfield}{areaoffield}\right)\times areaofwell\times dilution}{infectionvolume}$$

      Note: For reference, the 10× lens of microscope in our lab has a field size of 0.27 mm², while the area size of a well for an 8-well chamber slide is 0.79 cm².

Figure. 3.

The schematic diagram of VRP plasmid

(Adapted from Agnihothram et al. 2018,¹⁹ J Virol).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Brilliant Violet (BV) 510 anti-mouse CD45	BioLegend	103138
APC/Cyanine 7 anti-mouse CD3e	BioLegend	100330
PE/Cyanine 5 anti-mouse CD4	BioLegend	100410
PE/Cyanine 7 anti-mouse CD8	BioLegend	100722
APC anti-mouse IFN-γ	BioLegend	505810
FITC anti-mouse TNF	BioLegend	506304
PE anti-mouse IL2	BioLegend	503808
FcγR blocker	BD	553141
FITC goat anti-mouse IgG	Abcam	Ab6785
Bacterial and virus strains
MERS-CoV (EMC2012 strain or equivalent)	Erasmus Medical Center, Rotterdam, Netherlands	N/A
Chemicals, peptides, and recombinant proteins
DMEM powder	Gibco	12100-046
DMEM solution	Gibco	11960-044
MEM	Gibco	M200500
RPMI	Gibco	11875119
FBS	Atlanta Biologicals	s11150
Penicillin/streptomycin	Gibco	15140-122
Na-pyruvate	Gibco	11360-070
NEAA	Gibco	11140-050
Na-bicarbonate	Gibco	25080-094
L-glutamine	Gibco	25030-081
ACK buffer	Gibco	A10492-01
1× PBS	Gibco	10010-023
1× PBS (Mg++ and Ca++)	Gibco	14040-133
Sucrose	Invitrogen	15503022
0.25% Trypsin-EDTA	Gibco	25200-056
Type 3 collagenase	Worthington Biochem	LS004180
DNase I	Worthington Biochem	LS006330
Formaldehyde solution	Sigma	47608-1L-F
Brefeldin A	Sigma	B6542
DMSO	Sigma	67-68-5
Agarose powder	Roche	11685678001
Acetone	Sigma	270725
Methanol	Sigma	34860
Crystal violet powder	Fisher	c581
Ketamine	Vetaket	NDV-59399-114-10
Xylazine	Anased	NDC-59399-111-50
Not I enzyme	Thermo Fisher Scientific	ER0591
T7 Transcription kit	Ambion	AM1334
Virex II 256	Virex	N/A
Micro-Chem	NCL	1839-95-2296
MERS-CoV-specific peptides	Bio-Synthesis	N/A
Experimental models: Organisms/strains
hDPP4-KI mice (C57BL/6J background) 8–10 weeks old, male or female	University of Iowa	N/A
Experimental models: Cell lines
Vero81	ATCC	CCL-81
BHK	ATCC	CCL-10
Software and algorithms
FlowJo software	BD	v10.8
Prism	GraphPad	8
Other
Live/dead cell kit	Invitrogen	L34964
Anti-fade buffer	Vector Laboratories	UX-93952-24
CountBright™ Absolute Counting Beads	Invitrogen	C36950
Cytofix™ Fixation Buffer X2	BD	BDB554655
Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit	BD	BDB554714
pVR-21 and plasmids encoding the 3526 capsid and E glycoproteins	Dr. Ralph Baric, University of North Carolina	N/A

Materials and equipment

Culture medium recipes

D10 media

Reagent	Final concentration	Amount
DMEM	N/A	500 mL
FBS	10%	60 mL
Penicillin/streptomycin	100 Units/mL/100 μg/mL	6 mL
Na pyruvate	0.67 mM	4 mL
NEAA	1×	6 mL
Na-bicarbonate	0.15%	12 mL
L-glutamine	4 mM	12 mL
Total	N/A	600 mL

Storage: 4°C, 2 weeks.

D2 mdium

Reagent	Final concentration	Amount
DMEM	N/A	500 mL
FBS	2%	11 mL
Penicillin/streptomycin	100 Units/mL/100 μg/mL	6 mL
Na pyruvate	0.67 mM	4 mL
NEAA	1×	6 mL
Na-bicarbonate	0.15%	12 mL
L-glutamine	4 mM	12 mL
Total	N/A	551 mL

Storage: 4°C, 2 weeks.

Complete MEM medium

Reagent	Final concentration	Amount
MEM	N/A	500 mL
FBS	10%	60 mL
Penicillin/streptomycin	100 Units/mL/100 μg/mL	6 mL
Na pyruvate	0.67 mM	4 mL
NEAA	1×	6 mL
Na-bicarbonate	0.15%	12 mL
L-glutamine	4 mM	12 mL
Total	N/A	600 mL

Storage: 4°C, 2 weeks.

Complete RPMI medium

Reagent	Final concentration	Amount
RPMI	N/A	500 mL
FBS	10%	60 mL
Penicillin/streptomycin	100 Units/mL/100 μg/mL	6 mL
Na pyruvate	0.67 mM	4 mL
NEAA	1×	6 mL
Na-bicarbonate	0.15%	12 mL
L-glutamine	4 mM	12 mL
Total	N/A	600 mL

Storage: 4°C, 2 weeks.

VRP preparation recipes

pVR21 Digestion mixture

Reagent	Final concentration	Amount
Buffer3	N/A	6.5 μL
100× BSA	1.1×	0.7 μL
Not I	8.57 Units/mL	2.0 μL
H2O	N/A	5.8 μL
pDNA	N/A	50.0 μL
Total	N/A	65.0 μL

Storage: prepare right before usage.

Transcription mixture

Reagent	Final concentration	Amount
pDNA	N/A	7.5 μL
10× Buffer	1×	3.0 μL
2× NTP/CAP	8.57 Units/mL	15.0 μL
Enzyme	N/A	3.0 μL
GTP	N/A	1.5 μL
Total	N/A	30.0 μL

Storage: prepare right before usage.

Other recipes

1% Crystal violet

Reagent	Final concentration	Amount
Crystal violet	1%	5 g
ddH2O	N/A	395 mL
Methanol	N/A	105 mL
Total	N/A	500 mL

Storage: 25°C, 3 months.

Mouse tissue digestion buffer

Reagent	Final concentration	Amount
type III Collagenase	3 mg/mL	1.5 g
DNase I	5 μg/mL	2.5 mg
1× PBS	N/A	500 mL
Total	N/A	500 mL

Storage: aliquot into 10 mL/tube, −20°C, 6 months.

Step-by-step method details

Immunization of hDPP4-KI mice with a sublethal dose of MERS-CoV

Timing: 3 min/mouse

In this section, we describe intranasal (i.n.) immunization of hDPP4-KI mice with a sublethal dose of live MERS-CoV on day 0. The 3R (replacement, reduction, and refinement) principle needs to be followed in all animal experiment design. Intraperitoneal immunization with live virus may also induce humoral and cellular immune responses to MERS-CoV but will be less efficient in generating airway-resident immune memory in recipients. Manipulations of mice for immunization are similar to those used for mouse infection described in MERS-CoV propagation and titration.

• 1.
  Immunization with a sublethal dose of live MERS-CoV (Day -28).

  Note: All of the following procedures need to be caried out in a ABSL3 lab wearing PPE with institutional permission.

  • a.
    Determination of immunization dose.

    Note: The dose of MERS-CoV used to immunize mice should result in moderate morbidity without causing death of mice. Based on the outcome of MERS-CoV virulence test described in MERS-CoV propagation and titration, the optimal dose used to immunize mice shall lead to a 10–20% loss of initial weight. The peak of weight loss usually occurs during 7–10 dpi and mice may start to regain their weight, responsiveness, and activity from 10 dpi.

    Note: To avoid inter-batches variation, the same batch of virus should be used to determine viral virulence and for immunization. Virus sequence should be verified.

    Note: To avoid the loss of viral titer and virulence caused by repeated freezing and thawing, all virus remaining after each experiment will be inactivated and discarded.

  • b.
    Preparation of hDPP4-KI mice.

    Note: Same as described in MERS-CoV propagation and titration.

  • c.
    Immunization of hDPP4-KI mice.

    Note: Same as described in MERS-CoV propagation and titration.

After returning mice to the original cages after they completely awake, monitor their weight and general condition 1–2 times per day. If humane endpoints (as described in Mers-CoV propagation and titration) are reached, although unlikely with the sublethal infection, mice will be euthanized by CO₂ inhalation followed by cervical dislocation.

Immunization of hDPP4-KI mice with 2 doses of VRP-MERS-S

Timing: 3 min/mouse

In this section, we describe i.n. immunization with 2 doses of VRP-MERS-S on day -70 and -42. Similarly, we highlight the important 3R considerations.

• 2.
  Immunization with VRP-MERS-S can be carried out in a ABSL1 laboratory with institutional permission.)

  • a.
    Preparation of hDPP4-KI mice is the same as described in MERS-CoV propagation and titration.
  • b.
    Immunization of hDPP4-KI mice is the same as described in MERS-CoV propagation and titration, except that 2 doses of VRP-MERS-S will be inoculated to hDPP4-KI mice, i.n., 25 μL /nostril, 50 μL total.

Note: Different from sublethal MERS-CoV immunization, mice receiving VRP-MERS-S are NOT expected to show any significant abnormal signs (such as substantial weight loss, irritation, hunch, and respiratory distress) after immunization.

Immune response assessment and virus challenge

Timing: Day -28 to 42 post re-challenge with MERS-CoV

In this section, we describe the assessment of virus-specific humoral (neutralizing antibody-mediated) and cellular (CD4 and CD8 T cell-mediated) immune responses at multiple time points after immunization with a sublethal dose of live MERS-CoV or two doses of VRP-MERS-S. In vivo challenge of a lethal dose of MERS-CoV will also be executed to verify the protective efficacy of immunizations. As described above, we highlight the important 3R considerations. All animal manipulations are carried out following the protocol approved by IACUC.

• 3.
  Determination of virus-specific neutralizing antibody in the serum of immunized mice (The following procedures may be carried out in ABSL3, or ABSL1 laboratory dependent on the immunization method. Nevertheless, PPE is needed for all manipulations.)

  • a.
    Collection of serum samples.

    • i.
      Check the general condition (activity, responsiveness, healthy fur) of mice and anesthetize them in an isoflurane chamber (0.5 lpm (liters per min) oxygen and 2 lpm isoflurane).

      Note: it usually takes 5 min to obtain complete anesthesia.

    • ii.
      Remove anesthetized mice from the chamber and restrain them with the non-dominant hand by grasping the loose skin over the shoulders and behind the ears; the skin should be taut over the mandible.
    • iii.
      After cleaning the face skin with 70% EtOH pad, puncture the facial vein with a 25-gauge needle or a lancet slightly behind the mandible, but in front of the ear canal.

      Note: A swift lancing motion should be used to puncture the vessel. Only the tip of the needle should enter the vessel to a shallow depth of 1–2 mm. Blood will flow immediately.

    • iv.
      Collect sample with capillary tubes or pipettman tips. When the sample has been collected (0.2 mL), apply gentle pressure to the blood collection site with a sterile gauze sponge until bleeding has stopped.

      Note: If facial punch is not allowed, tail cutting can be an optional method to collect blood sample. The tail tips (2 mm) of mice will be cut with sterile scissors under complete anesthesia and blood obtained. The wound then needs to be tightly pressed to stop any active bleeding and sterilized with 70% EtOH. The wound needs to be monitored daily until complete healing occurs. If infection of the wound is observed, the animal needs to be treated with antibiotics or euthanized as per institutional guidelines.

    • v.
      The animal may be returned to their home cage after completely awakening.

      Note: The recovery of mice from isoflurane anesthesia usually takes 2–3 min. To avoid irritation and stress of animals, manipulation needs to be swift and gentle. The procedure needs to be paused if the animal wakes up during the sample collection. Repeat the procedure to collect the sample from the vein on the other side of the face if necessary. At each time point, avoid repeated punctures at the same sites to reduce the risk of contamination. Sample collection will be carried out at a minimum interval of 2 days to allow wound healing.

    • vi.
      After the manipulation is finished, monitor the general condition and wound daily. An anti-inflammatory (meloxicam, 5 mg/kg, daily) or analgesic (buprenorphine SR, 0.5–1 mg/kg, every 48 h) treatment may be applied as necessary.
  • b.
    Titration of serum virus-specific antibody (PRNT₅₀ assay).

    • i.
      Place collected whole blood samples in 1.5 mL centrifuge tubes at 25°C for 4 h, to separate serum by precipitation.
    • ii.
      Centrifuge samples at 4,500× g for 5 min. Transfer the supernatant (serum) into a new clean 1.5 mL centrifuge tube and centrifuge at 4,500× g for 5 min.
    • iii.
      Transfer the clarified serum into a new screw cap tube or aliquot serum into multiple tubes per need (0.2 mL whole blood usually generates 0.05 mL serum).

      Note: Store serum in a −80°C freezer. Avoid repeated freezing and thawing, which may impair the efficacy of antibody function.

    • iv.
      Dilute serum with serum-free DMEM (we generally start our serial dilutions at a 1:10 dilution, which results in a final concentration of 1:20 after mixing with virus).
    • v.
      Mix diluted serum with MERS-CoV virus (diluted with serum-free DMEM at 400 PFU/mL) at a volume ratio of 1:1. Incubate the mixture in a 37°C incubator for 1 h.
    • vi.
      Remove the media from Vero 81 plates (prepared as described in MERS-CoV propagation and titration) and rinse cells with pre-warmed 1× PBS.
    • vii.
      After removing PBS by aspiration, add the mixtures of serum and virus into plates at 0.2 mL/well and make triplicates for each mixture.

      Note: Always leave 1 column (3 wells) for positive control (mixture of virus with verified serum) and 2 columns for negative controls (virus alone and the mixture of virus with serum harvested from naive mice).

    • viii.
      Return plates to the 37°C incubator for 1 h with gently shaking every 15 min.
    • ix.
      Remove the mixtures and rinse cells with pre-warmed 1× PBS.
    • x.
      Quickly add prepared 0.6% DMEM agarose into plates, 0.5 mL/well.
    • xi.
      Leave the plates in the Biosafety cabinet at 25°C, until the agarose solidifies.
    • xii.
      Add 0.5 mL D2 medium overlay to the agarose-DMEM in each well.
    • xiii.
      Return plates to the 37°C, 5% CO₂ incubator.
    • xiv.
      On day 3, read plaques (visualized by Crystal violet staining like Figure 2) as described in MERS-CoV propagation and titration.
    • xv.
      The titer of neutralizing antibody is calculated by regression analysis.²⁰

      Note: The titration of samples to be examined usually starts at a 1:20 dilution to minimize their non-specific effects on virus replication. Serum titers <1:20 are regarded as non-virus specific or non-neutralizing.

• 4.
  Determination of virus-specific T cell responses in the lung and the spleen of immunized mice (The following procedures may be carried out in ABSL3 or ABSL1 lab dependent on the immunization methods. Nevertheless, the PPE will be needed for all manipulations.)

  • a.
    Collection of lung and spleen samples.

    • i.
      Anesthetize immunized mice with 100 mgkg^-1/10 mgkg^-1 Ketamine/Xylazine via i.p. injection in 0.2 mL volume using a 1 mL insulin syringe.
    • ii.
      Examine the responsiveness of animals by pinching the tail or limb to assess anesthesia and fix animals to the operation platform in a supine position.
    • iii.
      Clean the abdominal skin with 70% EtOH pad and cut the skin open with scissors or scalpels to expose the subcutaneous tissue.
    • iv.
      Lift the tissue with forceps and cut the thoracic and peritoneal cavity open with sterile scissors.

      Note: Pay attention not to puncture the heart and other organs.

    • v.
      After bleeding by cardiac puncture, insert a 25-G needle into the right ventricle and perfuse with 10–20 mL cold 1× PBS.

      Note: The PBS should be administered under constant pressure slowly. High pressure may damage the normal structure of the lung.

      Note: The mice will die in minutes after thoracic cavity is opened due to the loss of negative pressure. Therefore, the perfusion needs to be carried out immediately after opening the thoracic cavity. A good perfusion results in inflated white lung lobes in healthy mice.

    • vi.
      Remove the lung and separate the lobes (total of 5), and the spleen with sterile scissors and plate the tissues in cold 1× PBS.
  • b.
    Homogenization of lung and spleen samples.

    • i.
      Rinse lung lobes and spleen with cold 1× PBS.
    • ii.
      Plate a 70 μm cell strainer in a 6-well plate, then add 5 mL cold 1× PBS into the well.
    • iii.
      Plate spleen in the strainer and homogenize with the cap of a sterile 1.5 mL centrifuge tube or the plunger of a 1 mL syringe.
    • iv.
      Remove the filtered cell suspension at the bottom of the well to a clean 15 mL tube.
    • v.
      Rinse the cell strainer with 5 mL cold 1× PBS and add PBS to cell suspension.
    • vi.
      Centrifuge the pooled samples at 300× g for 10 min, at 4°C and resuspend the pellets in Ammonium-Chloride-Potassium (ACK) buffer (GIBCO, A10492-01) to remove red blood cells (RBC) (4°C, 5 min).
    • vii.
      Centrifuge cells at 300× g for 10 min, at 4°C, discard the supernatant and re- suspend the cell pellets with pre-warmed 10% FBS-RPMI, followed by cell counting under microscope.
    • viii.
      Cut lung lobes into 2–5 mm³ pieces with sterile scissors and place in a 70 μm cell strainer. Insert the strainer in a 6-well plate and immerse lung pieces in 5 mL pre-warmed 3 mg/mL type III Collagenase (NC9405360) /5 μg/mL DNase I (LS006342) (Sigma Worthington) cocktail.
    • ix.
      Leave the plate in a 37°C incubator for 30 min.
    • x.
      Homogenize the lung pieces in the cell strainer with the cap of a sterile 1.5 mL centrifuge tube or the plunger of a 1 mL syringe.
    • xi.
      Remove the filtered cell suspension into a clean 15 mL tube. Rinse the strainer with 5 mL cold 1× PBS and pool wash with cells. Centrifuge at 300× g for 10 min at 4°C and remove RBC with ACK buffer, 5 min, at 4°C.
    • xii.
      Centrifuge at 300× g for 10 min, at 4°C and discard the supernatant and re-suspend the cell pellets with pre-warmed 10% FBS-RPMI, followed by cell counting using a microscope.
  • c.
    Determination of virus-specific T cell responses by in vitro peptides or peptide pools stimulation followed by flow cytometry assay.

    • i.
      Dilute single cell suspension of the lung and spleen with pre-warmed 10% FBS-RPMI at a concentration of 20×10⁶/mL and plate into round-bottom 96-well plates, 0.1 mL/well.
    • ii.
      Dissolve MERS-CoV peptides or peptide pools (MERS-CoV-specific immunodominant peptides are predicted using online programs and synthesized by Bio-Synthesis, 85% purity)²¹ in Dimethyl sulfoxide (DMSO).

      Note: The stock solutions should be prepared at a concentration of 10–100 mM and stored at a −80°C freezer (stable for 2 years).

    • iii.
      Prepare peptides or peptide pools working solutions immediately before use. Thaw 1 vial of stock solution at 25°C and dilute with 10% FBS-RPMI to a concentration of 4 μM.

      Note: The final concentration of DMSO in cell culture system should be no more than 1:1000.

    • iv.
      Add 0.1 mL of working solution into each well pre-seeded with 0.1 mL of lung or spleen cells and gently mix by up and down with a 200 μl pipetman.
    • v.
      Place the 96-well plates in a 37°C, 5% CO₂ incubator, and culture for 6 h.
    • vi.
      To block the secretion of induced cytokines, Brefeldin A (BFA) (Invitrogen, B6542) is added into the culture at a final concentration of 10 μg/mL for the final 4 hr of incubation.
    • vii.
      To quantify cell numbers, CountBright™ Absolute Counting Beads (10⁶ beads/ml, Invitrogen, C36950) are added into each well, 5–10 μl /well at the start of culture.
    • viii.
      centrifuge plates at 300× g for 10 min at 4°C and remove the culture medium. Re-suspend cell pellets with 0.2 mL cold serum-free RPMI. Add 10 μg/mL FcγR blocker (BD, 553141) into each well according to the manufacturer’s manual.
    • ix.
      Centrifuge the plates at 300× g for 10min at 4°C and remove the supernatants.
    • x.
      Resuspend cell pellets with 0.1 mL cold serum-free RPMI containing fluorescence-labeled antibodies targeting surface markers (BV510-conjugated rat anti-mouse CD45 (clone 30-F11), APC-Cy7-conjugated hamster anti-mouse CD3 (clone 145-2C11), PerCP Cy5.5-conjugated rat anti-mouse CD4 (clone GK1.5), and PE-Cy7-conjugated rat anti-mouse CD8 (clone 53–6.7). Live/Dead cell dye (Invitrogen, L34964) is also added at the concentration according to the manufacturer’s manual. Stain cells in the dark at 25°C for 15 min. All antibodies used here are purchased from Biolegend.
    • xi.
      Add 0.1 mL 1× PBS/well and centrifuge the plates at 300× g for 10 min at 4°C.
    • xii.
      Remove the supernatant and re-suspend cell pellets with 0.1 mL Cytofix/Cytoperm buffer (BD, BDB554714). Keep the plates in the dark at 4°C for 15 min.
    • xiii.
      Add Perm/Wash buffer (BD, BDB554714), 0.1 mL/well and centrifuge the plates at 300× g for 10 min at 4°C.
    • xiv.
      Stain cells with fluorescence-labeled antibodies targeting intracellular molecules (such as APC-conjugated rat anti-mouse IFN-γ (clone XMG1.2), FITC-conjugated rat anti-mouse TNF (clone MP6-XT22), PE-conjugated rat anti-mouse IL-2 (clone JES6-5H4, all from Biolegend)) in dark at 4°C for 30 min.
    • xv.
      Add 0.1 mL Perm/Wash buffer/well and centrifuge the plates at 300× g for 10 min at 4°C.
    • xvi.
      Remove the supernatant and resuspend cells with 0.2 mL 1× PBS or running buffer.
    • xvii.
      Transfer cell suspension into flow cytometry tubes and perform flow cytometry assay (BD FACSVerse).
    • xviii.
      Analyze data obtained from flow cytometry with Flowjo software (v10.8, strategy and expected outcomes are shown in Figure 4).

      Note: The selection of antibody and conjugated fluorescein needs to be carefully designed and reviewed with the guidance of experienced staff. The overlap of fluorescence may significantly affect the outcomes and needs to be solved by compensation adjustments. Due to length restrictions, the details of flow cytometry will not be discussed in this protocol.

• 5.
  Challenge immunized mice with a lethal dose of MERS-CoV (day 0). All of the following procedures need to be carried out in ABSL3 with PPE with institutional permission).

  • a.
    Preparations of hDPP4-KI mice are same as described in MERS-CoV propagation and titration.
  • b.
    Re-challenge of hDPP4-KI mice is same as described in MERS-CoV propagation and titration.

Note: Infected mice may start to lose weight from 2 dpi and may show systemic and respiratory symptoms (as described in MERS-CoV propagation and titration) starting at 4–5 dpi. Water (Nepa Nectar) and food will be placed at the bottom of the cages to be reached easily. Intraperitoneal injection with 1× PBS may be necessary to treat dehydration. The death of mice mostly occurs between 7–10 dpi. Mice will be euthanized once humane endpoints (described in MERS-CoV propagation and titration) are reached.

Figure. 4.

Gating strategy for T cell flow cytometry and expected intracellular staining outcomes

(A) The gating of CD4 and CD8 T cells from lung single cell suspension of MERS-CoV-treated mice. Beads: CountBright™ Absolute Counting Beads.

(B) The intracellular IFN-gamma, TNF and IL-2 expression in CD8 T cells isolated from mock- or MERS-CoV-treated mice was determined by flow cytometry after in vitro stimulating lung single cell suspension with SARS-CoV-2 S protein peptide pool at 7 dpi.

All surviving animals will be euthanized by CO₂ inhalation followed by cervical dislocation at 14 dpi, while some mice may be euthanized at indicated time points for tissue harvesting as required.

Expected outcomes

Both one sublethal dose of MERS-CoV and two doses of VRP-MERS-S immunization induce protective immune memory responses to viral antigens of MERS-CoV. Compared to control groups (naïve mice or DMEM-treated mice), immunized mice should be protected from lethal re-challenge of MERS-CoV (characterized by limited or no weight loss and ameliorated respiratory or systemic symptoms). Besides clinical outcomes, lung histological examination (hematoxylin and eosin staining) and determination of cytokine or chemokine profiles in lung and peripheral blood (by quantitative polymerase chain reaction (Q-PCR), enzyme-linked immunosorbent assay (ELISA) or Luminex assay) may also be used as readouts.

Sublethal doses of MERS-CoV immunization should induce both humoral (virus-specific neutralizing antibody) and cellular immune memory (CD4 and CD8 T cell specific for MERS-CoV peptides or peptide pools). In addition to measuring PRNT₅₀, virus-specific and total antibody production in immunized mice can be determined by ELISA, which will measure the total amount of virus-specific antibody (including both neutralizing and non-neutralizing antibodies). Additionally, determination of antibody levels (especially those of virus-specific IgA) in the nasal and bronchoalveolar lavage of immunized mice will demonstrate mucosal immune responses induced by immunization. As for cellular immunity, the distribution, and the accumulation of virus-specific and total CD4 or CD8 T cells can be identified by virus-specific cytokine expression after peptide stimulation as described above or using viral peptide-loaded tetramers, followed by flow cytometry.

VRP-MERS-S immunization will also induce virus-specific neutralizing antibody and CD4/CD8 T cell responses. Compared to sublethal dose of live MERS-CoV, VRP-MERS-S is relatively weak in inducing immune responses due to the lack of in vivo replication and less stimulatory effects, which makes boosting necessary for the formation of effective immune memory. On the other hand, since VRP-MERS-S contains only the spike protein of MERS-CoV, memory CD4 or CD8 T cell response can only be detected by stimulation with spike protein-derived peptides or peptide pools or corresponding tetramers. Therefore, peptides or peptide pools of other MERS-CoV proteins, such as those of membrane protein and nucleoprotein, can be used as unrelated antigen control in the T cell response assessment measured by cytokine expression as described in step 4.

Quantification and statistical analysis

A Student’s t test or 1-way ANOVA with Turkey’s post hoc correction is used to analyze differences in mean values between groups. Multiple regression analysis is used to test the repeated measurements between different groups adjusted for time after infection or immunization. Differences in mortality are analyzed using Kaplan-Meier log-rank survival tests. All results are analyzed using Microsoft Excel and GraphPad Prism 8 and are expressed as Mean ± SEM. P values of less than 0.05 were considered statistically significant. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Limitations

In the present protocol, immune memory responses are induced by a sublethal dose of live MERS-CoV or 2 doses of VRP-MERS-S.

Immunization with a sublethal dose of live MERS-CoV needs to be performed in an ABSL-3 lab. After immunization, the mice need to be kept in the ABSL-3 lab until re-challenge or euthanasia for tissue collection. In the present protocol, we determine the immune memory responses within -28 to 42 days post re-challenge. To determine the long-term outcomes of immunization, the mice will need to be housed in the ABSL-3 lab for an extended period. The availability of the ABSL-3 lab may be limited and the costs of experiments in ABSL-3 are higher compared to usual animal facilities. In addition, the training of qualified users of ABSL-3 is costly and time-consuming. Immunization with VRP-MERS-S and the determination of virus-specific immune responses in an ex vivo system (such as T cell responses assayed using MERS-CoV peptides or peptide pool stimulation) can be accomplished in an ABSL-1 lab. However, immunized mice need to be challenged in the ABSL-3 lab.

The effects of immunization are usually dose dependent. To induce immune memory with a sublethal dose of live MERS-CoV, immunization needs to be capable of inducing potent immune responses without killing mice. In the present protocol, 10–20% weight loss is used as the index to identify the immunization dose. However, the outcome of each single experiment may vary, especially when different batches of virus are used. Common problems include weight loss (generally <5%) or occasionally, more severe disease including animal death. To improve the efficacy of immunization, the dose test described in MERS-CoV propagation and titration needs to be carefully designed and executed with sufficient animal numbers (n ≥ 8/dose). Likewise, the dose of VRP-MERS-S used for immunization needs to be optimized. Due to their relatively weak stimulatory capacity and the lack of clinical symptoms after immunization, we use as highest dose as possible, although 1×10⁷ VRP/dose is usually sufficient to induce potent immune memory responses. As discussed in expected outcomes, the antibody titer determined by PRNT₅₀ represents only the quantity of virus-specific neutralizing antibody, while other assays such as ELISA may be needed to determine the full spectrum of antibodies induced by immunization. Virus-specific T cell responses may vary according to the quality of peptides or peptide pools produced by different manufacturers. A reliable provider can be the key to the expected outcomes of experiments.

CoV infection also results in age-dependent disease severity. Therefore, the immunization dose and challenge dose used in this protocol (subjects: 8–10 weeks old mice) may be too high for older mice. To investigate the immune memory responses in older mice (8–10-months-old middle-aged mice or >18-months-old aged mice), the in vivo virulence of MERS-CoV will needs to be re-assessed as described in MERS-CoV propagation and titration. Finally, male hDPP4 mice are usually more susceptible to MERS-CoV infection compared to age-matched female mice. The dose used for immunization and challenge obtained from male mice may not be optimal for female mice, while the underlying mechanisms of this sex differences remain to be clarified.

Troubleshooting

Problem 1

Potential side effects of anti-inflammatory drugs used in step 3 on immune responses.

Potential solution

• •The best way to control side effects of anti-inflammatory drugs is to optimize the animal host and experimental environment, as well as improve experimental skills, to minimize or avoid the use of anti-inflammatory drugs.
• •If anti-inflammatory drugs are used, appropriate control groups (such as animals receiving sham manipulations and anti-inflammatory drugs) need to be set up for monitoring potential side effects of anti-inflammatory drugs.
• •Veterinarians may be consulted on the type, dose, and treating route to minimize or avoid potential side effects of anti-inflammatory drugs.

Problem 2

Purity of virus.

Potential solution

• •In this protocol, MERS-CoV is propagated in tissue cell cultures. Although centrifugation is used to purify virus, it is impossible to completely remove cellular components. Additional purification with 0.2–0.4 μm filters or by ultra-centrifugation (as described in VRP-MERS-S preparation) is useful in improving the purity of virus.
• •Although unlikely, remaining cellular components or culture medium components may affect the outcomes of immunization and infection. To minimize these effects, medium of uninfected cell culture can be used to treat control mice (uninfected or mock-treated).

Problem 3

No virus-specific neutralizing antibody or virus-specific T cell immune responses are detected, with or without protection from MERS-CoV lethal challenge.

Potential solution

• •If both ex vivo and in vivo assays of immune memory responses fail, it generally means that the immunization is unsuccessful. Titration of MERS-CoV and of VRP-MERS-S needs to be repeated in cells and in mice. The stability of stocks stored at −80°C and of working solutions (always kept on ice and diluted based on correct calculation) needs to be verified as well.
• •If immune responses are detected but are weaker than expected, the most likely reason is the suboptimal condition of virus or peptides or inappropriate manipulations with the working solutions.
• •If in vivo challenge shows protection whereas ex vivo assays show low responses, the protocols and performance of ex vivo assays need to be reviewed.
• •Although unlikely, if in vivo challenge fails to show protection whereas ex vivo assays show expected outcomes, MERS-CoV used for challenge need to be assessed for potential mutations using sequence analysis.
• •Other effector or memory T cell markers such as APC-conjugated rat anti-mouse CD44 (clone IM7), FITC-conjugated hamster anti-mouse CD69 (clone H1.2F3), PE-conjugated rat anti-mouse CCR7 (clone 4B12), Pacific blue-conjugated rat anti-mouse CD62L (clone MEL-14), can be used to identify memory T cell subsets.²² However, these markers do not characterize virus specificity of T cells.

Problem 4

Intra-group variations are high.

Potential solution

• •To control intra-group variation, the SOP must be carefully followed.
• •The stock and manipulation of virus need to be reviewed.
• •The manipulation of mice, especially the immunization and infection of mice, needs to be reviewed. Inappropriate anesthetization or inoculation of virus will greatly affect the efficacy of immunization and infection. Make sure that mice are fully anesthetized before administering virus or immunogen to minimize the loss of inocula caused by choking.
• •Finally, the lab staff need to be well trained to ensure reproducible results.

Problem 5

The background noise of the flow data is high, or the positive signal is low.

Potential solution

• •The determination of T cell responses using peptides or peptide pools stimulation followed by flow cytometry assay needs to be piloted in preliminary experiments.
• •The strength of noise and signal generated by flow cytometry can be influenced by multiple factors. The time point of sample collection and the manipulation of samples represent the major variables that affect the responsiveness of T cells after stimulation. After immunization and challenge, the associated inflammation (particularly in those immunized with live MERS-CoV) may increase the expression of cytokines by T cells, especially lung T cells, in an antigen-independent manner. To minimize by-stander effects, appropriate controls (such as unrelated peptides or phorbol myristate acetate (PMA)/ionomycin stimulation) are needed to normalize the baseline of cytokine expression.
• •The isolation and stimulation of T cells need to be optimized. T cells need to be manipulated carefully and gently for optimal responses. The health status of T cells determined by Live/dead cell dying kit as described in step 4 is an important reference to predict their responsiveness. In addition, as discussed in limitations, the peptides or peptide pools provided by different manufacturers may significantly affect the outcomes.
• •Another potential solution to the signal problem is the optimization of the flow cytometry assay, including selection of fluorescence-labeled antibodies, compensation of fluorescence signals, and refinement of data analysis.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to, and will be provided by the lead contact, Stanley Perlman (stanley-perlman@uiowa.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate/analyze datasets/code.